Vector particles for targetng cd34+ cells

ABSTRACT

The present invention relates to a vector particle for transferring biological material into cells, wherein said vector particle comprises at least: —a first protein which comprises the transmembrane and extracellular domains of the feline endogenous RD114 virus envelope glycoprotein, and —a second protein which comprises a ligand of the c-Kit receptor.

The present invention relates to vector particles intended for thespecific delivery of biological material to cells.

For the correction by gene therapy of many inherited or acquired defectsof the hematopoietic system, the therapeutic gene must be delivered tocells able both to self-renew and to differentiate into allhematopoietic lineages. As such, these gene therapies must be targetedto the “right” cells, i.e. hematopoietic stem cells (HSCs), withoutmodifying their properties. The population of choice for targeting HSCsis constituted of CD34⁺ progenitor cells, which are particularlyenriched in these stem cells. However, CD34⁺ cells only represent 0.001%of the total blood cells for instance. Accordingly, to avoid thecumbersome steps of cell extraction, culture in the presence of multiplegrowth factors or transduction adjuvants, and infusion into the patient,the vector particles have to display a very high specificity towardsCD34⁺ cells, in order to allow transduction of CD34⁺ cells innon-purified bodily samples, such as blood samples, or to ensure anefficient in vivo transduction of CD34⁺ cells despite dilution of thevector particles.

Thus, Sandrin et al. (2002) Blood 100:823-832 have devised SimianImmunodeficiency Virus (SIV)-derived vector particles which display achimeric envelope glycoprotein, RDTR, constituted of the fusion of thetransmembrane and extracellular domains of the feline endogenous RD114virus envelope glycoprotein and the cytoplasmic domain of the MurineLeukemia Virus-A envelope glycoprotein. Such vector particles are alsodisclosed in WO 03/91442. When using a transduction adjuvant, such asRetroNectin®, the transduction rate obtained using vector particlesdisplaying the chimeric RDTR protein is of approximately the same rateas that observed with SIV-derived vector particles displaying theVesicular Stomatitis Virus (VSV) G envelope glycoprotein. However, inthe absence of transduction adjuvant, the RDTR vector particles exhibita much lower transduction of isolated CD34⁺ cells than vectorsdisplaying the VSV-G glycoprotein. Besides, no particular selectivitytowards CD34⁺ cells has been shown to be associated to RDTR, sincevector particles displaying this chimeric protein transduce CD34⁺ cellsand peripheral blood lymphocytes with approximately the same efficiency.

In another attempt at targeting CD34⁺ cells, Verhoeyen et al. (2005)Blood 106:3386-3395 have devised HIV-1-derived vector particles whichdisplay the VSV-G envelope glycoprotein and so-called early actingcytokines, namely Thrombopoietin (TPO) and Stem Cell Factor (SCF). Theauthors have thus shown that these vector particles provided forefficient transduction of isolated CD34⁺ cells. However, no targetingspecificity of these vector particles could be evidenced.

Accordingly, it is an object of the present invention to provide vectorparticles which are more efficient than those of the prior art atspecifically targeting CD34⁺ cells.

SUMMARY OF THE INVENTION

The present invention arises from the discovery, by the inventors, thatthe co-display of RDTR and SCF on HIV-derived vector particles hadunexpected synergic effects on the efficiency and the specificity oftransduction of CD34⁺ cells.

Advantageously, such vector particles are not dependant on RetroNectin®to achieve transduction, can effect efficient transduction at lowdosage, and are capable to transduce CD34⁺ cells in fresh whole blood.

Thus, the present invention relates to a vector particle fortransferring biological material into cells, wherein said vectorparticle comprises at least:

-   -   a first protein which comprises the transmembrane and        extracellular domains of the feline endogenous RD114 virus        envelope glycoprotein, and    -   a second protein which comprises a ligand of the c-Kit receptor.

The present invention also relates to the use of (i) a first nucleicacid comprising a sequence encoding a first protein as defined above andof (ii) a second nucleic acid comprising a sequence encoding a secondprotein as defined above, for preparing a vector particle fortransferring biological material into cells and in particular forpreparing a vector particle as defined above.

The present invention also relates to a method for preparing a vectorparticle for transferring biological material into cells and inparticular for preparing a vector particle as defined above, wherein (i)a first nucleic acid comprising a sequence encoding a first protein asdefined above and (ii) a second nucleic acid comprising a sequenceencoding a second protein as defined above, are transferred in aproducer cell, and the vector particle is recovered from said producercell.

The present invention also relates to a medicament comprising a vectorparticle as defined above as active ingredient.

The present invention also relates to a method for treating anindividual in need of gene therapy, wherein a therapeutically effectiveamount of a vector particle as defined above is administered to theindividual.

The present invention further relates to the use of a vector particle asdefined above, for transferring the biological material into cells exvivo.

The present invention also relates to a method for preparing cellsintended for treating an individual, wherein cells to be administered tothe individual are contacted with a vector particle as defined above.

The present invention also relates to a method for treating anindividual in need of gene therapy, wherein in a first step cells to beadministered to the individual are contacted with a vector particle asdefined above and in a second step said cells are administered to theindividual.

The present invention also relates to a protein represented by SEQ IDNO: 4.

The present invention also relates to a nucleic acid encoding a proteinof sequence SEQ ID NO: 4.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 represents the percentage of CD34⁺ cells (vertical axis)transduced by GFP-encoding HIV-derived vector particles displaying RDTRonly, in the presence of recombinant TPO (10 ng/ml) or recombinant SCF(50 ng/ml), or vector particles displaying RDTR and TPOHA, or RDTR andSCFHA, in the presence (widely hatched bars) or absence (closely hatchedbars) of RetroNectin®.

FIG. 2 represents the percentage of CD34⁺ cells (vertical axis)transduced by GFP-encoding HIV-derived vector particles displaying RDTRonly, in the presence of recombinant TPO (10 ng/ml) or recombinant SCF(50 ng/ml), or vector particles displaying RDTR and TPOHA, or RDTR andSCFHA, at a Multiplicity Of Infection (M.O.I.) of 10 (widely hatchedbars), 2 (white bars) or 0.2 (closely hatched bars) as determined onHeLa cells.

FIG. 3 represents the percentage of GFP expressing cells (vertical axis)present in a PBMC population isolated from cord blood transduced byGFP-encoding HIV-derived vector particles displaying RDTR only in thepresence of recombinant SCF (50 ng/ml), RDTR and SCFHA, VSV-G only inthe presence of recombinant SCF (50 ng/ml), or VSV-G and SCFHA, whereinthe cells are CD34⁺ cells (closely hatched bars) or CD3⁺ cells (widelyhatched bars).

FIG. 4 represents the percentage (vertical axis) of GFP expressing CD34⁺cells or CD3⁺ cells present in whole cord blood transduced byGFP-encoding HIV-derived vector particles displaying RDTR only in thepresence of recombinant SCF (50 ng/ml) (first bar), RDTR and SCFHA(second bar), VSV-G only and recombinant SCF (50 ng/ml) (third bar), orVSV-G and SCFHA (fourth bar).

FIG. 5 represents the analysis by fluorescence-activated cell sorter(FAGS) of the transduction (GFP⁺) of total human cells in the bonemarrow. The three histograms show respectively the results obtained onthree different injected mice. The cells were sorted according to hCD45expression (hCD45⁺, vertical axis) and GFP expression (GFP⁺, horizontalaxis).

FIG. 6 represents the analysis by FACS of the transduction (GFP⁺) ofearly progenitors (hCD34⁺), myeloid progenitors (hCD13⁺), monocytes(hCD14⁺) and pre- and pro B-cells (hCD19⁺) in the bone marrow. The firsthistogram shows the results obtained with cells sorted according tohCD34 expression (hCD34⁺, vertical axis) and GFP expression (GFP⁺,horizontal axis). The second histogram shows the results obtained withcells sorted according to hCD13 expression (hCD13⁺, vertical axis) andGFP expression (horizontal axis). The third histogram shows the resultsobtained with cells sorted according to hCD14 expression (hCD14⁺,vertical axis) and GFP expression (horizontal axis). The fourthhistogram shows the results obtained with cells sorted according tohCD19 expression (hCD19⁺, vertical axis) and GFP expression (horizontalaxis).

FIG. 7 represents the analysis by FACS of the transduction (GFP⁺) ofhuman thymocytes in the thymus. The cells were sorted according to hCD45expression (hCD45⁺, vertical axis) and GFP expression (GFP⁺, horizontalaxis).

FIG. 8 represents the analysis by FACS of the transduction (GFP⁺) ofB-cells (hCD19⁺) and T-cells (hCD3⁺) in the peripheral blood. The firsthistogram shows the results obtained with cells sorted according tohCD19 expression (hCD19⁺, vertical axis) and GFP expression (GFP⁺,horizontal axis). The second histogram shows the results obtained withcells sorted according to hCD3 expression (hCD3⁺, vertical axis) and GFPexpression (horizontal axis).

FIG. 9 represents the analysis by FACS of the transduction (GFP⁺) ofhuman splenocytes (hCD45⁺), B-cells (hCD19⁺) and T-cells (hCD3⁺) in thespleen. The first histogram shows the results obtained with cells sortedaccording to hCD45 expression (hCD45⁺, vertical axis) and GFP expression(GFP⁺, horizontal axis). The second histogram shows the results obtainedwith cells sorted according to hCD19 expression (hCD19⁺, vertical axis)and GFP expression (horizontal axis). The third histogram shows theresults obtained with cells sorted according to hCD3 expression (hCD3⁺,vertical axis) and GFP expression (horizontal axis).

DETAILED DESCRIPTION OF THE INVENTION

As intended herein, “vector particle” denotes any particle liable todisplay the first protein and the second protein at its surface and toreversibly bind to a biological material.

It is preferred that such a vector particle is a viral vector particle,in particular a lentiviral vector particle, such as a lentiviral vectorparticle selected from the group consisting of Human ImmunodeficiencyVirus (HIV), e.g. HIV-1 or HIV-2, and Simian Immunodeficiency Virus(SW).

Lentiviral vector particles are well-known to the man skilled in the artand are notably described in Naldini et al. (2000) Adv. Virus Res.55:599-609 and Negre et al. (2002) Biochimie 84:1161-1171. Usually,lentiviral vector particles according to the invention comprise at leastthe following components: (i) an envelope component, which isconstituted of a phospholipidic bilayer associated to envelope proteins,wherein the envelope proteins comprise at least the above-defined firstand second proteins, said envelope surrounding (ii) a core component,constituted of the association of a gag protein, said core itselfsurrounding (iii) genome components, usually constituted of ribonucleicacids (RNA), and (iv) an enzyme component (pol). The biological materialcan be present within the envelope, within the core and/or within thegenome components.

Lentiviral vector particles can be readily prepared by the man skilledin the art, for example by following the general guidance provided bySandrin et al. (2002) Blood 100:823-832. Briefly, the lentiviral vectorparticles may be generated by co-expressing the packaging elements (i.e.the core and enzyme components), the genome component and the envelopecomponent in a so-called producer cell, e.g. 293T human embryonic kidneycells. Typically from three to four plasmids may be employed, but thenumber may be greater depending upon the degree to which the lentiviralcomponents are broken up into separate units.

Generally, one plasmid encodes the core (gag) and enzymatic (pol)lentiviral components of the vector particle. The origin of the gag andpol genes gives its name to the lentiviral vector particle. For instancethe expression “HIV-1-derived vector particle” usually indicates thatthe gag and pol genes of the vector particle are those of HIV-1. Thisplasmid is termed the packaging plasmid. One or several other plasmidsencode the proteins which are part of the envelope. In the present casethese plasmids may notably encode the first and the second protein. Aswill be clear to one of skill in the art, the above defined first andsecond nucleic acid may be either distinct or fused. Yet another plasmidencodes the genome.

As intended herein the expression “biological material” relates to oneor more compounds liable to alter the structure and/or the function of acell. Within the context of the present invention, it is preferred thatthe biological material is one or more nucleic acids, which in the caseof lentiviral vector particles may be comprised within the genome of thevector particle. The genome typically comprises the one or more nucleicacids, preferably linked to genetic elements necessary for theirexpression in the target cell, such as promoters and terminators,flanked by cis-acting elements necessary for the inclusion of the genomein the core element, its reverse transcription into deoxyribonucleicacid (DNA), the import of the retrotranscribed genome into the nucleusof the target cell and the integration of the retrotranscribed genomewithin the genome of the target cell.

As intended herein the recipient cells for the biological material to betransferred, or target cells, relate to any cell liable to be bound bythe above-defined vector particle. Where the vector particle is alentiviral vector particle the target cell relates to any cell liable tobe transduced by the vector particle. These cells usually express thec-Kit receptor which binds to the c-Kit ligand of the first protein. Assuch, the cells preferably targeted by the vector particle of theinvention are CD34⁺ cells, in particular human CD34⁺ cells, and moreparticularly Hematopoietic Stem Cells (HSCs), notably human HSCs.

As intended herein “transferring” relates to the capacity of the vectorparticle to initially deliver the biological material to the membrane orthe cytoplasm of the target cell, upon being bound to the target cell.After delivery, the biological material can be translocated to othercompartment of the cell.

The feline endogenous RD114 virus envelope glycoprotein is notablydescribed in Cosset et al. (1995) J. Virol. 69:7430-7436. By way ofexample, the RD114 virus envelope glycoprotein corresponds to GenBankaccession number X87829. Portions of RD114 corresponding to thetransmembrane and extracellular domains can be readily identified by theman skilled in the art.

As intended herein, the expression “transmembrane and extracellulardomains of the feline endogenous RD114 virus envelope glycoprotein”relates to transmembrane and extracellular domains of a natural felineendogenous RD114 virus envelope glycoprotein or to any mutant thereofderived therefrom by deletion, insertion or substitution of one orseveral amino acids, provided that said mutant presents essentially thesame properties as the transmembrane and extracellular domains of thenatural feline endogenous RD114 virus envelope glycoprotein from whichit derives.

As intended herein, a mutant will be said to present essentially thesame properties as the transmembrane and extracellular domains of anatural feline endogenous RD114 virus envelope glycoprotein from whichit derives, if, when replacing the transmembrane and extracellulardomains of a natural feline endogenous RD114 virus envelope glycoproteinin a reference vector particle according to the invention carrying afirst protein of sequence SEQ ID NO: 2 and a second protein of sequenceSEQ ID NO: 4, the mutant-carrying vector particle presents at least 30%,preferably at least 50%, more preferably at least 75%, of thetransduction of CD34⁺ cells which can be observed with the referencevector particle. Preferably, the transduction conditions are those setforth in Example 2.

By way of example, the transmembrane and extracellular domains of thefeline endogenous RD114 virus envelope glycoprotein are represented bySEQ ID NO: 5.

Preferably, the first protein comprises or consists in a fusion of thetransmembrane and extracellular domains of the feline endogenous RD114virus envelope glycoprotein and the cytoplasmic domain of a retroviralenvelope glycoprotein. In this fusion it is preferred that theC-terminus of the transmembrane domain of RD114 is fused to theN-terminus of the cytoplasmic domain of a retroviral envelopeglycoprotein.

More preferably, the first protein comprises or consists in a fusion ofthe transmembrane and extracellular domains of the feline endogenousRD114 virus envelope glycoprotein and the cytoplasmic domain of theMurine Leukemia Virus-A envelope glycoprotein. In this fusion it ispreferred that the C-terminus of the transmembrane domain of RD114 isfused to the N-terminus of the cytoplasmic domain of MLV-A envelopeglycoprotein.

The Murine Leukemia Virus-A envelope glycoprotein is notably describedin Ott et al. (1990) J. Virol. 64:757-766. Preferably, the MurineLeukemia Virus-A envelope glycoprotein is that of strain 4070A. Theportion of Murine Leukemia Virus-A envelope glycoprotein correspondingto the intracellular domain can be readily identified by the man skilledin the art. By way of example the intracellular domain of MurineLeukemia Virus-A envelope glycoprotein is represented by SEQ ID NO: 6.

Most preferably, the first protein is represented by SEQ ID NO: 2 and isin particular encoded by SEQ ID NO: 1. A preferred plasmid forexpressing the first protein in a producer cell is represented by SEQ IDNO: 11.

The c-Kit receptor is well known to the man skilled in the art. It isnotably described by Ashman (1999) Int. J. Biochem. Cell. Biol.31:1037-1051. By way of example, the human c-Kit receptor is encoded bySEQ ID NO: 8. Accordingly, it is well within the reach of the manskilled in the art to identify, design or select ligands of the c-Kitreceptor.

The natural ligand of the c-Kit receptor is the Stem Cell Factor (SCF)cytokine. The SCF cytokine is notably described by Ashman (1999) Int. J.Biochem. Cell. Biol. 31:1037-1051. As such, in the above-defined vectorparticle, the ligand of the c-Kit receptor is preferably the SCFcytokine. As intended herein the expression SCF cytokine relates to anatural SCF cytokine or to any mutant of a natural SCF cytokine derivedfrom said natural SCF by deletion, insertion or substitution of one orseveral amino acids, wherein said mutant retains the ability of thenatural SCF cytokine to bind to the c-Kit receptor. Preferably, the SCFcytokine is the human SCF cytokine. By way of example the human SCFcytokine corresponds to GenBank reference number P21583. It is mostpreferred that the SCF cytokine used herein is deprived of its signalpeptide and of its transmembrane and cytoplasmic domain (i.e. only theextracellular domain of the SCF cytokine is used), e.g. as representedby SEQ ID NO: 9.

More preferably, the second protein of the above-defined vector particlecomprises or consists in a fusion of the SCF cytokine and (i) theN-terminal domain of an hemagglutinin glycoprotein, or (ii) a retroviralenvelope glycoprotein. In this fusion it is preferred that theC-terminus of SCF is fused to the N-terminus of the N-terminal domain ofthe hemagglutinin glycoprotein or to the N-terminus of the retroviralenvelope glycoprotein.

Preferably, the hemagglutinin glycoprotein is that of an influenzavirus, more preferably of the Fowl Plague Virus.

Preferably, the N-terminal domain of the hemagglutinin glycoproteincomprises or consists in the contiguous amino acids from the N-terminusof the glycoprotein to the C-terminus of the HA1 subunit.

The subunit structure of the hemagglutinin glycoprotein is well known toone of skill in the art. The Fowl Plaque Virus hemagglutinin is notablydescribed in Hatziioannou et al. (1998) J. Virol. 72:5313-5317.

By way of example the N-terminal domain of the Fowl Plague Virushemagglutinin is represented by SEQ ID NO: 10.

Preferably, in the second protein, the retroviral envelope glycoproteinis Murine Leukemia Virus-A envelope glycoprotein.

As will be apparent to anyone of skill in the art, the second proteinmay also preferably comprise a signal peptide intended for promotingendoplasmic reticulum translocation of the second protein. In certaincases the signal peptide can be cleaved during or after insertion in thetargeted membrane. Such signal peptides are well known to the manskilled in the art and can be found, for example, at the extremities ofmembrane proteins. By way of example the signal peptide can be that ofthe Murine Leukemia Virus-A envelope glycoprotein, which can berepresented by SEQ ID NO: 7.

Thus, the second protein preferably comprises or consists in a fusion ofthe SCF cytokine, the N-terminal domain of an hemagglutininglycoprotein, and a signal peptide. In this fusion it is preferred thatthe C-terminus of the signal peptide is fused to the N-terminus of SCF,and that the C-terminus of SCF is fused to the N-terminus of theN-terminal domain of the hemagglutinin glycoprotein.

When the second protein comprises or consists in a fusion of SCF and aretroviral envelope glycoprotein, it is preferred that the C-terminus ofSCF is fused to the N-terminus of the retroviral envelope glycoproteindeprived of its signal peptide, and that the N-terminus of SCF is fusedto the C-terminus of a signal peptide as defined above, which ispreferably the signal peptide of the retroviral envelope glycoprotein towhich it is fused.

Most preferably, the second protein is represented by SEQ ID NO: 4 andis in particular encoded by SEQ ID NO: 3. A preferred plasmid forexpressing the first protein in a producer cell is represented by SEQ IDNO: 12.

In a particular embodiment of the above-defined vector particle, thefirst protein is represented by SEQ ID NO: 2 and the second protein isrepresented by SEQ ID NO: 4.

In another particular embodiment, the second protein as defined above isfused to the first protein as defined above. Preferably, when the firstand second proteins are fused, the second protein consists of a SCFcytokine, optionally fused to a signal peptide as defined above. Morepreferably, when the first and second protein are fused, the C-terminusof a signal peptide is fused to the N-terminus of a SCF cytokine, theC-terminus of the SCF cytokine is fused to the N-terminus of theextracellular domain of RD114, and the C-terminus of the transmembranedomain of RD114 is fused to the N-terminus of the cytoplasmic domain ofa retroviral envelope glycoprotein.

The present invention also relates to the fused first and secondproteins as defined above and to the nucleic acids which comprisesequences encoding them.

In another particular embodiment, the above-defined vector particle doesnot comprise the Vesicular Stomatitis Virus (VSV) G envelopeglycoprotein.

The VSV-G envelope glycoprotein is notably described in Yee et al.(1994) Methods Cell Biol. 43:99-112. By way of example the VSV-Genvelope glycoprotein is represented by SEQ ID NO: 13.

As is apparent from the foregoing, the above-defined vector particle canbe used for the in vivo or ex vivo transfer of biological material tocells, in particular to CD34⁺ cells, and among them to HSCs.

Accordingly, the vector particle is particularly indicated for treatinghematopoietic cells-related diseases either by direct administration ofthe vector particle to the individual afflicted by such a disease, or byadministering cells, in particular cells originating from the individualafflicted by such a disease, which have been contacted ex vivo with thevector particle.

In this frame, it is preferred that the vector particle is a lentiviralvector particle as defined above and/or that the target cells aretransduced by one or more nucleic acids, preferably intended fortreating the disease.

The vector particle would thus be indicated for treating myelosupressionand neutropenias which may be caused as a result of chemotherapy,immunosupressive therapy, infections such as AIDS, genetic disorders ofhematopoietic cells, cancers and the like.

Exemplary genetic disorders of hematopoietic cells that are contemplatedinclude sickle cell anemia, thalassemias, hemaglobinopathies, Glanzmannthrombasthenia, lysosomal storage disorders (such as Fabry disease,Gaucher disease, Niemann-Pick disease, and Wiskott-Aldrich syndrome),severe combined immunodeficiency syndromes (SCID), as well as diseasesresulting from the lack of systemic production of a secreted protein,for example, coagulation factor VIII and/or IX.

In such cases, one would desire to transfer one or more nucleic acidssuch as globin genes, hematopoietic growth factors, which includeerythropoietin (EPO), the interleukins (especially Interleukin-1,Interleukin-2, Interleukin-3, Interleukin-6, Interleukin-12, etc.) andthe colony-stimulating factors (such as granulocyte colony-stimulatingfactor, granulocyte/macrophage colony-stimulating factor, or stem-cellcolony-stimulating factor), the platelet-specific integrin αllbβ,multidrug resistance genes, the gp91 or gp 47 genes which are defectivein patients with chronic granulomatous disease (CGD), antiviral genesrendering cells resistant to infections with pathogens such as humanimmunodeficiency virus, genes coding for blood coagulation factors VIIIor IX which are mutated in hemophiliacs, ligands involved in Tcell-mediated immune responses such as T cell antigen receptors, B cellantigen receptors (immunoglobulins), the interleukin receptor common γchain, a combination of both T and B cell antigen receptors alone and/orin combination with single chain antibodies (ScFv), IL2, IL12, TNF,gamma interferon, CTLA4, B7 and the like, genes expressed in tumor cellssuch as Melana, MAGE genes (such as MAGE-1, MAGE-3), P198, P1A, gp100etc.

Exemplary cancers are those of hematopoietic origin, for example,arising from myeloid, lymphoid or erythroid lineages, or precursor cellsthereof. Exemplary myeloid disorders include, but are not limited to,acute promyeloid leukemia (APML), acute myelogenous leukemia (AML) andchronic myelogenous leukemia (CML), Lymphoid malignancies which may betreated using a vector particle as defined above include, but are notlimited to acute lymphoblastic leukemia (ALL) which includes B-lineageALL and T-lineage ALL, chronic lymphocytic leukemia (CLL),prolymphocytic leukemia (PLL), hairy cell leukemia (HLL) andWaldenstrom's macroglobulinemia (WM). Additional forms of malignantlymphomas contemplated as candidates for treatment utilizing thelentiviral vector particles of the present invention include, but arenot limited to non-Hodgkin lymphoma and variants thereof, peripheralT-cell lymphomas, adult T-cell leukemia/lymphoma (ATL), cutaneous T-celllymphoma (CTCL), large granular lymphocytic leukemia (LGF) and Hodgkin'sdisease.

Where the vector particle is used as a medicament and is administered toan individual in a therapeutic method, administration through theintravenous route or by the medullar route, in particular the femur orhumerus medullar route, is preferred. For intravenous administration aunit dose from about 5·10⁸ to about 10⁹ vector particles as definedabove can be used, whereas for medullar administration a unit dose fromabout 10⁸ to about 5·10⁸ vector particles as defined above can be used.

Where the vector particle is used ex vivo the vector particle can becontacted, preferably in vitro, either with isolated or purified cells,such as CD34⁺ cells, or with non-purified bodily samples.

The cells can be isolated or purified from various tissues, inparticular taken from the individual, such as blood, in particular cordblood, or bone marrow.

Non-purified bodily samples can originate from the individual to betreated, and notably comprise blood samples, in particular whole cordblood samples.

The quantity of vector particle to be used for ex vivo transfers ofbiological material is for example from about 10⁷ to about 5·10⁷ forabout 10⁶ total white blood cells (where the cells to be transduced arecomprised in total white blood cells from a whole blood sample).

EXAMPLES Example 1 Production of Lentiviral Vector Particles (LVs)

The inventors displayed two early acting cytokines, Thrombopoietin (TPO)and Stem Cell Factor (SCF), on a lentiviral vector particle (LV)surface.

A TPO truncated form of 171-amino acid long, shown to have a 3-foldhigher biological activity than wild-type TPO, was fused to theN-terminus of the influenza hemagglutinin (HA) glycoprotein to formTPOHA. The second cytokine, SCF, was also fused to the N-terminus of HAglycoprotein to form SCFHA (SEQ ID NO: 4), which efficientlyincorporates on LVs.

Since these chimeric HA glycoproteins demonstrated a reducedinfectivity, an additional fusion competent glycoprotein wasco-expressed. A chimeric feline endogenous RD114 virus envelopeglycoprotein was chosen, in which the cytoplasmic tail of RD114 wasexchanged for that of Murine Leukemia Virus-A (MLV-A) env glycoproteinresulting in a mutant RDTR (SEQ ID NO: 2), that allows highincorporation onto HIV as well as SIV vector particles (Sandrin et al.(2002) Blood 100:823-832).

Thus, a transfection protocol was optimized to co-display SCFHA or TPOHAwith RDTR on HIV-derived lentiviral vector particles.

Briefly, 2.5·10⁶ 293T cells were seeded the day before transfection in10 cm plates in a final volume of 10 ml DMEM. The next day these cellswere cotransfected with an HIV or SIV gag-pol construct (8.6 μg) withthe lentiviral gene transfer vector particle (8.6 μg) and twoglycoprotein-encoding constructs selected from: a) VSV-G (1.5 μg) (SEQID NO: 14) or RDTR (SEQ ID NO: 11) (7 μg) and b) TPOHA (SEQ ID NO: 15)or c) SCFHA (SEQ ID NO: 12) (1.5 μg), using the Clontechcalcium-phosphate transfection system. 4 μg of a neuraminidase-encodingplasmid was also co-transfected to allow efficient release of vectorparticle from the producer cell since the HA (SCFHA and TPOHA) envelopeotherwise binds the vector particles to the producer cells because ofthe expression of sialic acid by the producer 293T cells. 15 h aftertransfection, the medium was replaced with 6 ml of fresh CellGro® medium(CellGenix) and 36 h after transfection, vector particles wereharvested, filtrated through 0.45 μm pore-sized membrane and stored at−80° C. The vector particles can be further concentrated viaultracentrifugation or polyethylene-glycol mediated concentration atlow-speed centrifugation.

Titers of 5·10⁵-10⁶ IU/ml were thus obtained, that were comparable toRDTR single pseudotyped vector particles.

Functional co-display of TPO on TPOHA/RDTR co-displaying vectorparticles was demonstrated on BAF3-Mpl cells, which are dependent on TPOfor survival and growth, essentially as described by Geddis et al.(2001) J. Biol. Chem. 276:34473-34479. Similarly, functional co-displayof SCF on SCFHA/RDTR vector particles was confirmed since they sustainedsurvival of BAF3-cKit cells which depend on SCF for survival (Bayle etal. (2004) J Biol Chem. 279:12249-12259), even at low multiplicity ofinfection (M.O.I.)

Example 2 Transduction of Isolated CD34⁺ Cells

The vector particles were first tested on the transduction of CD34⁺cells isolated from human cord blood (CB). CB CD34⁺ cells are veryimmature hematopoietic cells containing hematopoietic stem cells.

Briefly, CD34⁺ cells were isolated by positive selection usinganti-CD34⁺ beads (Miltenyi Biotech) from cord blood and were cultured onuncoated or RetroNectin® (Takara) coated plates. Subsequently, the cellswere incubated with Green Fluorescent Protein (GFP) encoding HIV derivedvector particles displaying RDTR, in the presence of human recombinantcytokines (TPO=10 ng/ml; SCF=50 ng/ml) (Preprotech, Rocky Hill, US), orco-displaying RDTR and TPOHA or RDTR and SCFHA, at a multiplicity ofinfection (M.O.I.) of 10, essentially as described by Verhoeyen et al.(2005) Blood 106:3386-3395.

As shown in FIG. 1, the resulting RDTR/SCFHA pseudotyped HIV vectorparticles were far more efficient in transducing cord blood-derivedCD34⁺ cells, than the LV pseudotyped with RDTR and TPOHA, or with RDTRonly in the presence of the corresponding cytokines in their solubleform. In addition, in contrast to the RDTRISCFHA pseudotyped HIV vectorparticles, the RDTR-only pseudotyped vector particles are completelydependent on RetroNectin® for the transduction of CD34⁺ cells(RetroNectin® is a chimeric peptide of human fibronectin produced inEscherichia coli which is thought to link vector particles and targetcells).

Thus, the above results indicate that an unexpected synergisticmechanism is taking place, between RDTR, allowing vector particle andcell fusion, and SCFHA, allowing specific binding and stimulation ofc-Kit⁺/CD34⁺ cells, which results in the high transduction efficiencyobserved.

Example 3 Multiplicity of Infection for CD34⁺ Cells

An important issue for the in vivo use of the vector particles of theinvention is that they should allow high transduction efficiency intoCD34⁺ cells even at very low vector particle dosage, since a systemicadministration of a therapeutic vector particle would result in animportant dilution of vector particle concentration. Thus, the inventorstested the minimal effective dosage of the vector particles according tothe invention.

Briefly, CD34⁺ cells were isolated by positive selection usinganti-CD34⁺ beads (Miltenyi Biotech) from cord blood and were cultured onuncoated culture plates (i.e. in the absence of RetroNectin®).Subsequently, the cells were incubated with Green Fluorescent Protein(GFP) encoding HIV derived vector particles displaying RDTR, in thepresence of human recombinant cytokines (TPO=10 ng/ml; SCF=50 ng/ml), orco-displaying RDTR and TPOHA or RDTR and SCFHA at a M.O.I. of 10, 2, or0.2, essentially as described by Verhoeyen et al. (2005) Blood106:3386-3395. At day 3 post initiation of transduction, cells wereevaluated for GFP expression by fluorescence-activated cell sorter(FACS).

As shown in FIG. 2, the RDTR/SCFHA vector particle of the inventionenabled a reduction of vector particle dosage to a M.O.I. of 0.2,without observing a significant drop in transduction efficiency of CD34⁺cells. Thus, a 50-fold decrease in RDTR/SCFHA vector particle dosageresulted on average only in a 1.4-fold reduction of CD34⁺ celltransduction. In contrast, the RDTR/TPOHA vector particle resulted in asignificantly lower CD34⁺ transduction when an M.O.I. of 0.2 was used.

Example 4 RDTR/SCFHA Targets Transduction to CD34⁺ Cells in a PeripheralMononuclear Blood Cell Population

A vector particle intended for in vivo gene therapy notably needs to behighly discriminative between target and non-target cells. Thus, afterhaving demonstrated the ability of the vector particle according to theinvention to transduce isolated CD34⁺ cells, its selectivity wasevaluated by adding vector particle to a whole peripheral bloodmononuclear cell (PBMC) population at low M.O.I. In this respect, it isimportant to highlight that no more than 1% CD34⁺ cells are contained insuch a population.

Briefly, PBMCs were isolated from fresh cord blood by ficol gradient, asis well-known to the man skilled in the art, and cultured in the absenceof RetroNectin®. Transduction of PBMCs was performed with GreenFluorescent Protein (GFP) encoding HIV derived vector particlesdisplaying RDTR or VSV-G in the presence of human rSCF (50 ng/ml), orco-displaying RDTR and SCFHA or VSV-G and SCFHA, without addingexogenous cytokines, at a M.O.I. of 0.2, essentially as described byVerhoeyen et al. (2005) Blood 106:3386-3395. At day 3 post initiation oftransduction, CD34⁺ and CD3⁺ cells were evaluated for GFP expression byfluorescence-activated cell sorter (FACS).

As shown in FIG. 3, the RDTR/SCFHA vector particle was able topreferentially target and transduce CD34⁺ target cells (up to 19%), insharp contrast to the vector particle pseudotyped with RDTR only, in thepresence of soluble SCF, which provided for no transduction at all, orto the VSV-G/SCFHA vector particle, which allowed a transduction levelof CD34⁺ cells of 5% at the most. Importantly, the RDTR/SCFHA vectorparticle allowed to transduce CD34⁺ cells within the PBMC population ata level equivalent to that obtained for the transduction of isolatedCD34⁺ cells (compare FIGS. 2 and 3). Furthermore, the T-cell population,which make up 80% of the whole PBMC population, was very poorlytransduced by the RDTR/SCFHA vector particle (FIG. 3). Worth noting,other cell lineages present in the PBMC population, such as monocytes,B-cells and NK-cells were not transduced at all.

Example 5 RDTR/SCFHA Targets Transduction to CD34⁺ Cells in In Vivo-LikeConditions

The inventors then devised conditions as close as possible to in vivosettings for targeting gene transfer into CD34⁺ cells. Thus, theinventors performed transduction of fresh total cord blood, whichcontains cells from each hematopoietic lineage: early progenitors,including Hematopoietic Stern Cells (HSGs), lymphocytes, monocytes, anderythrocytes. This allows, (i) evaluation of targeted gene transfer inthe CD34⁺ cells population, which represents only 0.001% of cells inwhole blood, and (ii) exposure of the vector particle to an active humancomplement system, an obstacle encountered by viral vector particles invivo.

Thus, fresh total cord blood (0.5 ml) was incubated with GFP encodingHIV vector particles pseudotyped with RDTR only or VSV-G only, in thepresence of soluble SCF (50 ng/ml), or co-displaying RDTR and SCFHA orVSV-G and SCFHA, without adding exogenous cytokines, at a M.O.I. of 0.01(calculated for the total amount of white and red blood cells present inthe blood sample). After 6-8 h incubation with the vector particles,total PBMCs were separated from the blood by a ficol gradient.

Subsequently, the CD34⁺ cells were isolated by positive selection usinganti-CD34⁺ beads (Miltenyi Biotech) and were further cultured in aserum-free medium in presence of soluble recombinant human SCF in orderto sustain survival until FACS analysis.

In order to reveal possible non-target gene transfer, after removal ofthe CD34⁺ cells, the residual PBMCs, consisting mainly of T-cells, werecultured in RPMI supplemented with anti-CD3 and anti-CD28 antibodies (BDPharmingen, Le Pont de Claix, France) and recombinant human IL-2(Preprotech Rocky Hill, US). This was done with a dual purpose: (i) toactivate T-cells in order to enable transduction, since the majority ofT-cells in the blood are in a quiescent state and accordingly are notpermissive to lentiviral transduction, and (ii) to sustain survival ofthese cells until analysis. Worth noting, very stringent conditions werethus used to reveal gene transfer in the non-target T-cell, which aremost probably never met in in vivo conditions. In other words theexperimental settings used most probably overestimate in vivonon-specific gene transduction of T-cell. At day 4 post initiation oftransduction, CD34⁺ and CD3⁺ cells were evaluated for GFP expression byfluorescence-activated cell sorter (FACS).

As shown in FIG. 4, the RDTR/SCFHA vector particle allowed atransduction of 4.5% CD34⁺ cells versus 0.4% for the VSV-G/SCFHA vectorparticle, while transduction with vector particles displaying VSV-G onlyor RDTR only is negligible. Thus, the RDTR/SCFHA vector particle is 10times more efficient than the VSV-G/SCFHA vector particle fortransducing CD34⁺ cells. In addition, the VSV-G/SCFHA vector particlereadily transduced the non-target T-cell population, resulting in an 1.8fold only selectivity for CD34⁺ cells transduction as compared toT-cell. In contrast, the RDTR vector particle demonstrates up to 95-foldselectivity for CD34⁺ cells as compared to T-cells. Thus, knowing thatonly 0.01% of the blood cells initially transduced are CD34⁺ cells andthat T-cells represent 1% of the blood cells, the RDTR/SCFHA vectorparticles efficiently target transduction to CD34⁺ cells.

As regards the low transduction efficiency achieved with the VSV-G/SCFHAvector particles, it might be due to the vector's susceptibility tohuman complement, which, as a consequence, would impair its use in vivo.

Example 6 RDTR/SCFHA Displaying LVs Allow Gene Transfer into hCD34⁺Cells In Vivo

The inventors assessed targeted gene transfer into HSCs by theRDTR/SCFHA vector particles in vivo in a humanized murine model.

Briefly, full and functional reconstitution of all human haematopoieticlineages including B and T-cells was achieved in newborn Rag2^(−/−);γc^(−/−) Balbc mice by injection with human umbilical cord blood (UCB)CD34⁺ cells. After 13 weeks of reconstitution the inventors detected onaverage 35% of human cells (hCD45⁺) in the bone marrow of these mice(FIG. 5) of which 5 to 15% expressed hCD34.

GFP-encoding RDTR/SCFHA vector particles were concentrated by low speedcentrifugation over a filtration column to obtain titers up to 5·10⁸IU/ml. 1·10⁵ infectious units of the RDTRISCFHA vector particles wereinjected into the femural bone marrow of the humanized mice from 13 weekof age on.

One week after the injection, three-colour marking was performed tomeasure GFP expression in the different haematopoietic lineages as wellas in the target hCD34⁺ cells in the bone marrow.

In the flushed bone marrow the inventors detected a transduction of upto 3% of the total human cells that had colonized the marrow of the mice(FIG. 5). Taking into account that a femur contains 1.5·10⁷ cells, theinventors administered a very low vector dose (MOI=0.006). However, aselective transduction of up to 3% of early human progenitors (hCD34⁺cells) and of 3% of the myeloid progenitors (hCD13⁺) in the BM wasdetected (FIG. 6). In contrast, monocytes and pre- and pro-B-cells weretransduced to a low extent (hCD14=0%; hCD19=0.2%). These results shouldbe explained by the fact, that one week after the injection,differentiation of hCD34⁺ cells, including transduced hCD34⁺ cells, intoearly progenitors such as hCD13⁺ myeloid progenitors and pre- and pro-Bcells may have already occurred.

Of utmost importance, the inventors verified in vivo escape of vectorsby analysing transduction of the other hematopoietic tissues. They didnot detect GFP⁺ human thymocytes (FIG. 7), nor transduction of humanCD19⁺ B-cells and CD3⁺T-cells in the blood stream of these intrafemuralinjected mice (FIG. 8). Additionally, they did not detect significantlevels of transduced B-cells (hCD19⁺ cells) and transduced T-cells inthe spleen (FIG. 9).

Summarizing, local administration of low doses of RDTR/SCFHA LV into theBM of humanized mice resulted in a selective transduction of hCD34⁺cells in vivo.

Sequence Identifiers Reference Table:

SEQ ID NO: Feature 1 Nucleic acid encoding a fusion of the transmembraneand extracellular domains of the feline endogenous RD114 virus envelopeglycoprotein and the cytoplasmic domain of MLV-A envelope glycoprotein 2Fusion of the transmembrane and extracellular domains of the felineendogenous RD114 virus envelope glycoprotein and the cytoplasmic domainof MLV-A envelope glycoprotein 3 Nucleic acid encoding a fusion of theSCF cytokine, the N-terminal domain of an influenza virus hemagglutininglycoprotein, and a signal peptide 4 Fusion of the SCF cytokine, theN-terminal domain of an influenza virus hemagglutinin glycoprotein, anda signal peptide 5 Transmembrane and extracellular domains of the felineendogenous RD114 virus envelope glycoprotein 6 Cytoplasmic domain ofMurine Leukemia Virus-A envelope glycoprotein 7 Signal peptide of theMurine Leukemia Virus-A envelope glycoprotein 8 Human c-Kit receptor 9Human SCF cytokine 10 N-terminal domain of the Fowl Plague Virushemagglutinin 11 Plasmid encoding the fusion protein of SEQ ID NO: 2 12Plasmid encoding the fusion protein of SEQ ID NO: 4 13 VSV-G envelopeglycoprotein 14 Plasmid encoding VSV-G 15 Plasmid encoding TPOHA

1. A vector particle for transferring biological material into cells,wherein said vector particle comprises at least: a first protein whichcomprises the transmembrane and extracellular domains of the felineendogenous RD114 virus envelope glycoprotein, and a second protein whichcomprises a ligand of the c-Kit receptor.
 2. The vector particleaccording to claim 1, wherein the ligand of the c-Kit receptor is theStem Cell Factor (SCF) cytokine.
 3. The vector particle according toclaim 1, wherein the vector particle does not comprise the VesicularStomatitis Virus (VSV) G envelope glycoprotein.
 4. The vector particleaccording to claim 1, wherein the vector particle is a lentiviral vectorparticle.
 5. The vector particle according to claim 4, wherein thelentiviral vector particle is selected from the group consisting of HIVand SIV.
 6. The vector particle according to claim 1, wherein the vectorparticle is intended for transferring biological material into CD34⁺cells.
 7. The vector particle according to claim 1, wherein thebiological material is one or more nucleic acids.
 8. The vector particleaccording to claim 1, wherein the first protein comprises or consists ina fusion of the transmembrane and extracellular domains of the felineendogenous RD114 virus envelope glycoprotein and the cytoplasmic domainof a retroviral envelope glycoprotein.
 9. The vector particle accordingto claim 8, wherein the cytoplasmic domain of a retroviral envelopeglycoprotein is that of Murine Leukemia Virus-A.
 10. The vector particleaccording to claim 1, wherein the first protein is represented by SEQ IDNO:
 2. 11. The vector particle according to claim 1, wherein the secondprotein comprises or consists in a fusion of a SCF cytokine and (i) theN-terminal domain of an hemagglutinin glycoprotein, or (ii) a retroviralenvelope glycoprotein.
 12. The vector particle according to claim 1,wherein the second protein comprises or consists in a fusion of a SCFcytokine and the N-terminal domain of an influenza virus hemagglutininglycoprotein.
 13. The vector particle according to claim 1, wherein thesecond protein is represented by SEQ ID NO:
 4. 14. The vector particleaccording to claim 1, wherein the first and the second proteins arefused.
 15. The vector particle according to claim 14, wherein the secondprotein consists of a SCF cytokine, optionally fused to an endoplasmicreticulum translocation signal peptide.
 16. The use of (i) a firstnucleic acid comprising a sequence encoding a first protein as definedin claim 1, and of (ii) a second nucleic acid comprising a sequenceencoding a second protein as in claim 1, for preparing a vector particlefor transferring biological material into cells.
 17. A medicamentcomprising a vector particle as defined in claim 1 as active ingredient.18. The use of a vector particle as defined in claim 1, for transferringthe biological material into cells ex vivo.
 19. The use according toclaim 18, wherein the cells are CD34⁺ cells.
 20. The use according toclaim 18, wherein the cells are comprised in a blood sample.
 21. Amethod for preparing cells intended for treating an individual, whereincells to be administered to the individual are contacted with a vectorparticle as defined in claim
 1. 22. The method according to claim 21,wherein the cells are CD34⁺ cells.
 23. The method according to claim 21,wherein the cells are comprised in a blood sample.
 24. The methodaccording to claim 21, wherein the cells are transduced by one or morenucleic acids transferred from the vector particle.
 25. A proteinrepresented by SEQ ID NO:
 4. 26. A nucleic acid encoding a proteinaccording to claim 25.